A chemiluminescent immunoassay kit for detecting quinolone or tetracycline antibiotic residues

Abstract

This application relates to the field of chemical detection, and in particular to a chemiluminescent immunoassay kit for detecting quinolone or tetracycline antibiotic residues. The kit comprises immunomagnetic beads formed from a superparamagnetic microparticle carrier and a specific monoclonal antibody, chemiluminescently labeled antigens, a matching detection instrument, and a standardized pretreatment reagent set. The immunomagnetic beads bind to the antibody via amide bonds formed through carboxyl activation. The labeled antigen is an acrid ester-labeled quinolone or tetracycline hapten. The characteristics of each component and the detection method steps are also described. This application achieves the technical effect of effectively detecting illegally added quinolone or tetracycline substances, with excellent performance of the superparamagnetic microparticle carrier, high antibody affinity, a reasonable method for preparing the labeled antigen, and a standardized pretreatment reagent set and suitable detection instrument and method, enabling quantitative analysis.

Description

Technical Field

[0001] This application relates to the field of chemical detection, and in particular to a chemiluminescent immunoassay kit for detecting quinolone or tetracycline antibiotic residues. Background Technology

[0003] In the regulatory testing phase, the existing testing technologies for veterinary drug preparations mainly include the following: Colloidal gold immunochromatography is a commonly used method due to its simplicity and ease of implementation at the grassroots level. High-performance liquid chromatography (HPLC) and high-resolution mass spectrometry (HMS) are also widely used, offering high accuracy and precise detection of components in veterinary drug preparations. However, these conventional testing technologies have significant limitations. While colloidal gold immunochromatography is simple to operate, its sensitivity is relatively low, making it difficult to detect low concentrations of residues. Although HPLC and HMS offer high accuracy, they suffer from complex sample pretreatment, long testing cycles, high equipment costs, and low throughput, failing to meet the rapid screening needs of grassroots supervision and enterprise self-inspection scenarios. Summary of the Invention

[0004] The purpose of this application is to overcome the above-mentioned technical problems and provide a chemiluminescent immunoassay kit for detecting quinolone or tetracycline antibiotic residues. A chemiluminescent immunoassay kit for detecting quinolone or tetracycline antibiotic residues, comprising: The invention comprises immunomagnetic beads formed by superparamagnetic microparticle carriers and specific monoclonal antibodies, chemiluminescent labeled antigens, and matching detection instruments, wherein the immunomagnetic beads bind to the antibody by forming amide bonds through carboxyl activation, and the labeled antigen is an acridinium ester-labeled quinolone or tetracycline hapten.

[0005] By adopting the above technical solution, immunomagnetic beads can effectively immobilize antibodies by forming amide bonds with them through carboxyl activation. Acrid ester-labeled quinolone or tetracycline haptens are used as labeled antigens. Combined with superparamagnetic microparticle carriers, immunomagnetic beads formed by specific monoclonal antibodies, and matching detection instruments, the detection of illegal quinolone or tetracycline additives can be achieved based on chemiluminescence immunoassay technology. It combines the high sensitivity of chemiluminescence with the high specificity of immunological methods, enabling rapid, high-throughput, and high-precision detection processes, meeting the needs of rapid screening in scenarios such as grassroots supervision and enterprise self-inspection.

[0006] Preferably, the surface modification layer of the superparamagnetic microparticle carrier contains carboxyl functional groups, the particle size of the microparticle carrier is 0.5-2 μm, the saturation magnetization is ≥50 emu / g, and the magnetic response separation time is ≤5 seconds.

[0007] By adopting the above technical solution, the carboxyl functional group-modified layer on the surface of the superparamagnetic microparticle carrier can be used to bind with antibodies to form immunomagnetic beads; the superparamagnetic microparticle carrier with a particle size of 0.5-2μm, saturation magnetization ≥50 emu / g, and magnetic response separation time ≤5 seconds can ensure that the carrier has good magnetic response performance, achieve rapid separation, improve detection efficiency, and at the same time ensure the stability and dispersibility of the carrier in the detection system.

[0008] Preferably, the immunomagnetic beads are coupled with antibodies via EDC / NHS activation, wherein the antibodies are IgG1 type monoclonal antibodies against quinolone or tetracycline antibiotics, and their affinity constant Ka ≥ 1 × 10⁻⁶. 9 L / mol.

[0009] By adopting the above technical solution and using the EDC / NHS activation method to conjugate antibodies, the activation efficiency can be improved; IgG1 type monoclonal antibodies against quinolone or tetracycline antibiotics with an affinity constant Ka ≥ 1 × 10⁻⁶ can be used. 9 L / mol ensures specific binding and high affinity between the antibody and the target, thereby improving the sensitivity and specificity of detection.

[0010] Preferably, the chemiluminescent labeled antigen is prepared by solid-phase synthesis, wherein the hapten molecule and the arm chain are connected by a C4 carbon chain, and the end of the arm chain forms a covalent bond with the acridine ester label through an NHS ester group.

[0011] By adopting the above technical solutions, the solid-phase synthesis method can ensure the accuracy and stability of chemiluminescent labeled antigen synthesis; the hapten molecule and the arm chain are connected by a C4 carbon chain, which can fully expose the antigen epitope and improve the binding ability with the antibody; the arm chain end forms a covalent bond with the acridine ester label through the NHS ester group, which can ensure the high efficiency and stability of acridine ester labeling, thereby improving the sensitivity and specificity of the kit detection.

[0012] Preferably, it also includes a standardized pretreatment reagent kit, the reagent kit comprising: A methanol-water mixture (8:2 by volume) is used for the extraction of powders / premixes. Acetonitrile solution is used for protein precipitation in oral solutions. A buffer solution containing BSA and Tween-20 was used to dilute the reaction system.

[0013] By adopting the above technical solutions, suitable pretreatment can be performed for veterinary drug samples of different dosage forms. For powders / premixes, a methanol-water mixture is used to enhance the dissolution of polar components. For oral liquids, acetonitrile is used to precipitate proteins to reduce turbidity interference. Buffer solutions containing BSA and Tween-20 are used to dilute the reaction system, which can shorten the extraction time to within 15 minutes, reduce the matrix interference rate to 5%-8%, and achieve a low-concentration residue recovery rate of 88%-92%. The cost of pretreatment per sample is reduced to 1.2 yuan, and the time taken is 18 minutes, meeting the needs of high-throughput screening.

[0014] Preferably, the detection instrument includes a magnetic separation device and a photomultiplier tube detection module, wherein the magnetic field strength applied by the magnetic separation device is ≥0.1T, and the operating voltage of the detection module is 800-1000V.

[0015] By adopting the above technical solution, the detection instrument is equipped with a magnetic separation device and a photomultiplier tube detection module. The magnetic field strength applied by the magnetic separation device is ≥0.1T, which can ensure the rapid separation of superparamagnetic microparticle carriers. The photomultiplier tube detection module operates at a voltage of 800-1000V, which can ensure accurate acquisition of chemiluminescence signals, thereby realizing accurate quantitative analysis of illegal additives such as quinolones or tetracyclines.

[0016] Preferably, the standardized pretreatment reagent kit further comprises: A direct dilution buffer for injectable formulations, containing a 0.22 μm filter membrane assembly; Inhibitors targeting complex matrix interference include polyethylene glycol 6000 and glycerin.

[0017] By adopting the above technical solution, the 0.22μm filter membrane component in the direct dilution buffer for injections can filter the injections and ensure the purity of the test samples; polyethylene glycol 6000 and glycerol, as inhibitors of complex matrix interference, can reduce the interference of complex matrix on the detection and improve the accuracy and reliability of the detection.

[0018] Preferably, the detection method includes the following steps: After the sample to be tested is treated with a matrix-matched extraction solution, it is incubated with immunomagnetic beads at 30-40℃ for 15-30 minutes. Solid-liquid separation is achieved by applying an external magnetic field. Adding a basic substrate triggers a chemiluminescent reaction. Quantitative analysis is performed by collecting luminescence intensity signals using a photomultiplier tube.

[0019] By adopting the above technical solution, rapid and accurate detection of illegal additives such as quinolones or tetracyclines can be achieved. The sample to be tested is treated with matrix-matched extraction solution, which can effectively dissolve the target analyte in different matrices. Incubation with immunomagnetic beads at a specific temperature and time facilitates the full binding of the target analyte with the antibody on the magnetic beads. Solid-liquid separation by an external magnetic field can quickly remove matrix impurities. The addition of an alkaline substrate triggers a chemiluminescence reaction, and the luminescence intensity signal is collected by a photomultiplier tube for quantitative analysis. The high sensitivity of chemiluminescence can be used to achieve accurate detection of the target analyte.

[0020] Preferably, the incubation step adopts a dynamic oscillation method, with an oscillation frequency of 50-150 r / min and an amplitude controlled within the range of 5-10 mm.

[0021] By adopting the above technical solution, a dynamic oscillation method is used in the incubation step, with an oscillation frequency of 50-150 r / min and an amplitude controlled within the range of 5-10 mm. This allows the immunomagnetic beads to fully contact the sample to be tested, promoting antigen-antibody reaction and improving detection efficiency and accuracy.

[0022] Preferably, the surface carboxyl group density of the superparamagnetic microparticle carrier is ≥150 μmol / g, and the dispersion coefficient (CV) of the carrier in PBS buffer is ≤10%.

[0023] By adopting the above technical solution, the high surface carboxyl group density of the superparamagnetic microparticle carrier can increase the binding sites with antibodies, thereby increasing the amount of antibody conjugation and helping to improve detection sensitivity; the low dispersion coefficient of the carrier in PBS buffer can ensure its uniform dispersion in solution, making the antigen-antibody reaction more complete, thereby improving the accuracy and stability of detection.

[0024] In summary, this application includes at least one of the following beneficial technical effects: 1. Immunomagnetic beads are formed by superparamagnetic microparticle carriers and specific monoclonal antibodies, which are then combined with acridinium ester-labeled antigens to achieve a detection sensitivity of 0.02 μg / kg, which is 25 times higher than that of traditional ELISA. This method can detect low concentrations of illegal additives in veterinary drugs. 2. By using the integrated technology of "one-step extraction-immunoenrichment" and standardized pretreatment reagent kit, the pretreatment time is shortened to 18 minutes and the pretreatment cost per sample is reduced to 1.2 yuan, meeting the needs of high-throughput screening. 3. The detection time has been shortened from the traditional 120 minutes to 35 minutes, with a throughput of 96 wells / batch, and 192 samples can be detected per hour. This achieves a three-dimensional improvement in "high sensitivity, high throughput, and speed," providing grassroots regulatory departments with a rapid screening tool and assisting in the accurate supervision of illegal additives. Attached Figure Description

[0025] Figure 1This is a side longitudinal section view of an example chemiluminescent plate of this application; Figure 2 This is a side cross-sectional view of the chemiluminescent plate of this application; Figure 3 This is a top view of the chemiluminescent plate of this application.

[0026] In the figure: 1. Outer frame support of the chemiluminescence plate; 2. Microporous strip for chemiluminescence reaction; 3. Reaction well. Detailed Implementation

[0027] The technical solutions in the embodiments of the present invention are further described in detail below. The described embodiments are merely possible technical implementations of the present invention, and are not limited thereto. Those skilled in the art can certainly combine the embodiments of the present invention to obtain other embodiments without creative effort, which are also within the protection scope of the present invention.

[0028] This application mainly uses a chemiluminescence immunoassay kit and detection method to achieve rapid and accurate detection of illegal additives in veterinary drug preparations. The following is a further detailed description of this application. Example

[0029] The chemiluminescent immunoassay kit for detecting illegally added quinolones or tetracyclines provided in this application includes immunomagnetic beads, chemiluminescent labeled antigens, a standardized pretreatment reagent kit, and a matching detection instrument, wherein: Immunomagnetic beads: formed by superparamagnetic microparticle carriers and specific monoclonal antibodies, which bind to the antibody through amide bonds formed by carboxyl activation; Chemiluminescent labeled antigens: acridinium ester-labeled quinolone or tetracycline haptens; Supporting testing instruments: Includes a magnetic separation device and a photomultiplier tube detection module, and is compatible with dedicated chemiluminescence plates (such as...). Figure 1-3 (As shown).

[0030] like Figure 1 (Side longitudinal section view) Figure 2 (Side cross-sectional view) and Figure 3As shown in the top view, the chemiluminescence plate includes an outer frame support 1, chemiluminescence reaction microporous strips 2, and reaction holes 3. The outer frame support 1 is made of chemically resistant polypropylene material and has a rectangular structure. It is used to fix the chemiluminescence reaction microporous strips 2 and ensure structural stability during the detection process. Its inner wall has slots for detachable installation of the microporous strips, facilitating batch replacement and cleaning. The chemiluminescence reaction microporous strips 2 are arranged parallel inside the outer frame support 1. Each microporous strip contains 12 independent reaction holes 3. The entire chemiluminescence plate has 8 microporous strips, forming a 96-hole layout (suitable for high-throughput detection requirements of 96 holes / batch). The reaction holes 3 have a conical bottom design with an inner diameter of 6mm and a depth of 10mm. The inner wall is hydrophilically treated to reduce liquid adhesion, ensure thorough mixing of the reaction system, and facilitate uniform magnetic field coverage by the magnetic separation device, improving solid-liquid separation efficiency.

[0031] The core principle behind the high sensitivity and specificity of this kit is as follows: the superparamagnetic microparticle carrier has a high specific surface area and rapid separation characteristics, which can improve antibody utilization and detection efficiency; the acridinium ester-labeled hapten can generate a stable chemiluminescent signal, which is convenient for quantitative analysis; and the 96-well layout and reaction well structure design of the chemiluminescent plate provide a standardized reaction site for antigen-antibody reaction and chemiluminescent signal generation, which, together with the matching detection instrument, enables accurate signal acquisition.

[0032] Specifically, immunomagnetic beads include superparamagnetic microparticle carriers and specific monoclonal antibodies: The surface modification layer of the superparamagnetic microparticle carrier contains carboxyl functional groups, with a particle size of 0.5-2 μm, a saturation magnetization of ≥50 emu / g, and a magnetic response separation time of ≤5 seconds. Fe3O4 magnetic beads (Fe3O4 magnetic cores are synthesized by co-precipitation and then carboxyl functional groups are introduced through surface modification) can be used, or other magnetic materials with similar magnetic properties and surface properties can be used as substitutes. Specific monoclonal antibodies are IgG1 type monoclonal antibodies against quinolone or tetracycline antibiotics, with an affinity constant Ka ≥ 1 × 10⁻⁶. 9 L / mol can be prepared through animal immunization, cell fusion and clonal screening (taking quinolone antibiotics as an example: select 6-week-old SPF grade BALB / c mice, immunize them with immunogen via intraperitoneal injection, screen mice with high serum titers after multiple booster immunizations, take spleen cells and fuse them with SP2 / 0 myeloma cells, and obtain hybridoma cell lines that stably secrete specific antibodies through clonal screening), or it can be prepared through genetic engineering technology. Immunomagnetic beads are coupled with antibodies via EDC / NHS activation: EDC activates the carboxyl group to generate an unstable O-acylisourea intermediate, and NHS converts it into a stable succinimide ester, which allows the antibody to form an amide bond with the carboxyl group on the surface of the magnetic beads, resulting in high coupling efficiency and stability.

[0033] Specifically, the chemiluminescent labeled antigen is prepared using a solid-phase synthesis method. The hapten molecule and its arm chain are linked via a C4 carbon chain, and the end of the arm chain forms a covalent bond with the acridine ester label via an NHS ester group. The design and synthesis of hapten molecules are crucial. AutoDockVina molecular simulation software can be used to analyze the molecular structure of quinolones or tetracyclines, locate the core region of the antigen epitope, and screen for the optimal hapten structure. During synthesis, Wang resin was used as a carrier, and the peptides were coupled step by step according to the standard peptide synthesis procedure. Acridinium ester labels have advantages such as high luminescence efficiency and fast luminescence speed, enabling labeled antigens to generate strong and stable light signals in chemiluminescence reactions. Other labels with similar luminescence properties can also be used as substitutes.

[0034] Specifically, the supporting testing instruments include a magnetic separation device and a photomultiplier tube testing module: The magnetic separation device applies a magnetic field strength ≥0.1T and adopts a "gradient magnetic field - short-time action" mode, which can quickly adsorb the immunomagnetic beads in the chemiluminescent reaction well 3 to achieve solid-liquid separation (in conjunction with the conical bottom design of the reaction well 3, the magnetic field can more evenly cover the magnetic bead aggregation area, shortening the separation time). The photomultiplier tube detection module operates at a voltage of 800-1000V. Its detection probe can be precisely aligned with each reaction hole 3 of the chemiluminescence plate, converting the chemiluminescence signal (430nm blue light) generated in the reaction hole into an electrical signal for detection and analysis, ensuring the sensitivity and accuracy of the detection. Other magnetic separation and detection devices with similar functions can also be used as alternatives, but it is necessary to ensure that they are compatible with the 96-well layout of the chemiluminescent plate.

[0035] The implementation principle of this embodiment is as follows: After pretreatment, the sample to be tested is added to the chemiluminescence reaction well 3 and incubated with immunomagnetic beads in the well. The target antigen binds to the antibody on the magnetic beads. The immunomagnetic beads at the bottom of the reaction well 3 are adsorbed by a magnetic separation device to remove matrix impurities. After adding an alkaline substrate, a chemiluminescence reaction occurs in the reaction well, and the photomultiplier tube detection module is aligned with the reaction well to collect the signal. The advantages of superparamagnetic microparticle carriers are used to improve antibody utilization and detection efficiency. The acrid ester-labeled hapten generates a stable luminescence signal. The standardized structure of the chemiluminescence plate ensures reaction consistency and high throughput requirements. The supporting instruments enable rapid separation and accurate detection, meeting the rapid screening needs in scenarios such as grassroots supervision and enterprise self-inspection. Compared with existing detection technologies, it has higher sensitivity, faster detection speed and lower cost. Example

[0036] The difference between this embodiment and the above embodiments is that the surface carboxyl group density of the superparamagnetic microparticle carrier is ≥150μmol / g, and the dispersion coefficient (CV) of the carrier in PBS buffer is ≤10%.

[0037] This design can further improve the coupling efficiency and stability of immunomagnetic beads, making the antibodies more evenly distributed on the surface of the magnetic beads and enhancing their binding ability with the target antigen. In the chemiluminescent reaction well 3, the evenly dispersed immunomagnetic beads can fully contact the sample to be tested, avoiding the problem of insufficient reaction caused by magnetic bead aggregation, thereby further improving the sensitivity and accuracy of detection.

[0038] The implementation principle of this embodiment is as follows: by optimizing the surface carboxyl group density and dispersion coefficient of the superparamagnetic microparticle carrier, the performance of the immunomagnetic beads is improved. Combined with the hydrophilic treatment and structural design of the reaction well 3 of the chemiluminescent plate, the antigen-antibody reaction is made more complete and stable in the well, thereby improving the detection effect of the entire kit. In practical applications, it can more effectively detect low concentrations of illegal additive residues, providing a more reliable guarantee for the quality supervision of veterinary drugs. Example

[0039] The kit in this embodiment, based on the above embodiment, also includes a standardized pretreatment reagent set, which comprises: Methanol-water mixture (volume ratio 8:2): used for the extraction of powders / premixes, enhancing the dissolution of polar components; Acetonitrile solution: used for protein precipitation in oral solutions to reduce turbidity interference; Buffer containing BSA and Tween-20: used for diluting the reaction system, providing a stable reaction environment, and reducing non-specific binding.

[0040] The implementation principle of this embodiment is as follows: the standardized pretreatment reagent set is designed with special extraction and processing methods for different dosage forms of veterinary drug preparations, which effectively removes matrix interference and improves the recovery rate of target substances; the processed sample is added to the chemiluminescence reaction well 3, which can avoid the interference of matrix impurities on the antigen-antibody reaction and luminescence signal in the well, providing a purer sample for detection, thereby improving the accuracy and reliability of the entire detection process and meeting the needs of different dosage form samples in actual detection. Compared with existing pretreatment methods, it has the advantages of simple operation, low cost and high efficiency. Example

[0041] Based on Example 3, the kit in this embodiment further includes the standardized pretreatment reagent set: Direct dilution buffer for injectables: Contains a 0.22μm filter membrane assembly, which can directly dilute injectables and filter impurities and microorganisms to ensure sample purity; Inhibitors targeting interference from complex matrices include polyethylene glycol 6000 and glycerol, which can reduce non-specific binding to complex matrices and improve detection stability at low temperatures.

[0042] The implementation principle of this embodiment is as follows: further improve the standardized pretreatment reagent set to provide a more effective treatment method for injectable and complex matrix samples; when the treated sample is reacted in the chemiluminescence reaction well 3, the impurity content is lower and the non-specific binding is less, which can better adapt to the detection of different types of veterinary drug preparations, improve the adaptability and accuracy of detection, and provide a more comprehensive solution for the quality detection of veterinary drug preparations. Example

[0043] The chemiluminescent immunoassay method for detecting illegally added quinolones or tetracyclines provided in this application includes the following steps: S1, Sample pretreatment and incubation: After treating the sample with a matrix-matched extraction solution, add it to reaction well 3 of the chemiluminescence plate, then add immunomagnetic beads, and incubate the chemiluminescence plate in a constant temperature shaking device at 30-40℃ for 15-30 minutes. Wherein: Powder / Premix: Extracted with a methanol-water mixture (volume ratio 8:2); Oral solution: Protein precipitation is performed using acetonitrile solution; Injection: Dilute with direct dilution buffer containing a 0.22 μm filter membrane assembly; The incubation adopts a dynamic oscillation method with an oscillation frequency of 50-150 r / min and an amplitude controlled within the range of 5-10 mm. (The outer frame support 1 of the chemiluminescence plate can effectively fix the microporous strip 2, avoiding displacement of the microporous strip or liquid leakage during oscillation. The hydrophilic treatment and conical bottom design of the reaction well 3 can promote the full mixing of the immunomagnetic beads and the sample, and improve the binding efficiency.)

[0044] S2, Solid-liquid separation: The incubated chemiluminescent plate is placed in a magnetic separation device. The magnetic separation device applies a magnetic field of ≥0.1T, with the magnetic field direction perpendicular to the bottom of the reaction well 3 of the chemiluminescent plate. The immunomagnetic beads quickly gather at the bottom of the reaction well 3 under the action of the magnetic field. After standing for 3-5 seconds, the supernatant is removed to achieve solid-liquid separation (the conical bottom design of the reaction well 3 can make the magnetic beads gather more concentrated, which is convenient for the complete removal of the supernatant and reduces residual interference).

[0045] S3, Chemiluminescence reaction: An alkaline substrate (a mixed solution of NaOH and H2O2) is added to each reaction well 3. The substrate reacts with the acridine ester-labeled antigen in the reaction well to produce 430nm blue light.

[0046] S4, Signal Acquisition and Quantitative Analysis: Move the chemiluminescence plate below the photomultiplier tube detection module. Align the probes of the detection module with each reaction well 3 in a preset order and acquire the luminescence intensity signal at a working voltage of 800-1000V. Convert the signal into an electrical signal using the instrument's built-in software and compare it with a standard curve (pre-plotted using a series of concentration standards on the same chemiluminescence plate) to calculate the concentration of the target antigen in the sample.

[0047] The implementation principle of this embodiment is as follows: The detection method of this embodiment combines chemiluminescence immunoassay technology, superparamagnetic microparticle carrier technology, and standardized chemiluminescence plate structure to achieve efficient detection through the following synergistic effects: 1. The 96-well layout of the chemiluminescence plate is adapted to high-throughput detection requirements, and can process 96 samples per batch. Combined with dynamic oscillation incubation, it can improve reaction efficiency. 2. The structural design of reaction well 3 is compatible with the magnetic separation device, which shortens the solid-liquid separation time and improves the efficiency of the detection process; 3. The photomultiplier tube detection module is precisely aligned with reaction hole 3 to ensure the accuracy of luminous signal acquisition; 4. Standardized pretreatment and optimized reaction conditions further ensure the sensitivity and specificity of the detection.

[0048] Compared with existing detection methods, this method reduces the detection time from 120 minutes to 35 minutes, achieves a throughput of 96 wells per batch, and can detect 192 samples per hour. At the same time, the detection sensitivity reaches 0.02 μg / kg, which is 25 times higher than that of traditional ELISA, providing effective technical support for the quality supervision of veterinary drug preparations.

[0049] The above are all preferred embodiments of this application, and are not intended to limit the scope of protection of this application. Therefore, all equivalent changes made in accordance with the structure, shape and principle of this application should be covered within the scope of protection of this application.

Claims

1. A residual quinolone or tetracycline antibiotic chemiluminescent immunoassay test kit, characterized by, Include: The invention comprises immunomagnetic beads formed by superparamagnetic microparticle carriers and specific monoclonal antibodies, chemiluminescent labeled antigens, and matching detection instruments, wherein the immunomagnetic beads bind to the antibody by forming amide bonds through carboxyl activation, and the labeled antigen is an acridinium ester-labeled quinolone or tetracycline hapten.

2. The reagent kit according to claim 1, characterized in that, The surface modification layer of the superparamagnetic microparticle carrier contains carboxyl functional groups. The particle size of the microparticle carrier is 0.5-2 μm, the saturation magnetization is ≥50 emu / g, and the magnetic response separation time is ≤5 seconds.

3. The reagent kit according to claim 2, characterized in that, The immunomagnetic beads are coupled with an antibody by EDC / NHS activation method, the antibody is IgG1 type monoclonal antibody against quinolone or tetracycline antibiotic, and the affinity constant Ka is greater than or equal to 1x10 9 L / mol.

4. The reagent kit according to claim 3, characterized in that, The chemiluminescent labeled antigen is prepared by solid-phase synthesis. Its hapten molecule and arm chain are connected by a C4 carbon chain, and the end of the arm chain forms a covalent bond with the acridine ester label through an NHS ester group.

5. The reagent kit according to claim 1, characterized in that, It also includes a standardized pretreatment reagent set, which comprises: A methanol-water mixture (8:2 by volume) is used for the extraction of powders / premixes. Acetonitrile solution is used for protein precipitation in oral solutions. A buffer solution containing BSA and Tween-20 was used to dilute the reaction system.

6. The reagent kit according to claim 1, characterized in that, The detection instrument includes a magnetic separation device and a photomultiplier tube detection module. The magnetic field strength applied by the magnetic separation device is ≥0.1T, and the operating voltage of the detection module is 800-1000V.

7. The reagent kit according to claim 5, characterized in that, The standardized pretreatment reagent kit also includes: A direct dilution buffer for injectable formulations, containing a 0.22 μm filter membrane assembly; Inhibitors targeting complex matrix interference include polyethylene glycol 6000 and glycerin.

8. The reagent kit according to claim 1, characterized in that, The detection method includes the following steps: After the sample to be tested is treated with a matrix-matched extraction solution, it is incubated with immunomagnetic beads at 30-40℃ for 15-30 minutes. Solid-liquid separation is achieved by applying an external magnetic field. Adding a basic substrate triggers a chemiluminescent reaction. Quantitative analysis is performed by collecting luminescence intensity signals using a photomultiplier tube.

9. The reagent kit according to claim 8, characterized in that, The incubation step employs a dynamic oscillation method, with an oscillation frequency of 50-150 r / min and an amplitude controlled within the range of 5-10 mm.

10. The reagent kit according to claim 2, characterized in that, The surface carboxyl group density of the superparamagnetic microparticle carrier is ≥150 μmol / g, and the dispersion coefficient (CV) of the carrier in PBS buffer is ≤10%.

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